substrate activation requires energy for the formation of covalent bond formation of the transition state complex

enzymes increase the rate of reaction,kcat, by decreasing the activation energy through either acid-base or electronic stabilization of the [ES]

Transition State Analog Inhibitors

highly potent and selective enzyme inhibitors because they mimic the three dimensional structure of the substrate transition state with pre-formed covalent bonds and thus require no energy of activation

bind enzymes a million fold tighter then substrate, and thus inhibitors can be administered at lower concentrations to avoid adverse drug toxicity

have high affinity(low Km)

Serine Protease

cleaves preproteins(zymogens) to make them active

pivotal role in blood coagulation(prothrombin) and digestion(trypsin, chymotrypsin and elastase)

hydrolyze peptide bonds on the carboxyl side of hydrophobic amino acids, Phe, tyr, trp and leu

dissociation of water to hydroxyl group(nucleophile) can attack the protein carbonyl group forming an oxyanion transition state complex

hydroxl addition to carbonyl froms a carboxylic groups, and the proton forms an amine

transmembrane proteins form a pore for ions that is either opened or closed in response to stimulus

may be voltage gated, ligand gated, or a regulator change in the intracellular domain(phosphorylation-gated and pressure gated)

Cystic Fibrosis transmembrane conductance regulator(CFTR) is an example of a ligand gated channel controlled by phosphorylation

Caveolae mediated transport

potocytosis

cave like structure in membrane

full of sphinoglipids

formed by caveolaen(cholesterol binding protein)

lipid raft?

ATP-Binding Cassette

Transporter Superfamily

bind ATP and use the energy to transport hydrophobic molecules across cell membranes

classified based on the homology in the ATP binding domains

consists of 7 mammalian subfamilies based on similarity in gene structure

chart in slides*

Fatty Acid Transporters

first fatty acids are freed from dietary fat by gastric lipases and transported across the apical membrane of the intestine actively by FATP4, FAT/CD36, FABPpm, or passively diffuse through lipid bilayer in enterocytes, FABPc facilitates fatty acid transport through the cytosol.

majority is re-esterfied to TAG and excreted into circulation as chylomicrons(CM)

can be activated by a G-protein that is dependent on low levels of AMP

GPrtotein also activated by AKT that is activated by insulin

mTOR activation signals to allow ribosome translation***

mTOR

TOR enzyme complex is a central controller for cell growth(accumulation of mass

metazoan(multi tissue) TOR is essential for growth during early development(deletion of the TOR gene is embryonic lethal in mammals)

TOR inclunce in mature or aging organisms is partly negative: unwanted cell proliferation; continued growth without division may lead to protein aggregates and abnormal proteins that may induce late life malfunction

also takes part in a negative feedback mech by inhibiting insulin receptor substrate

mTORC1 can also activate Grb10 that inhibits the insulin like growth factor receptor

Medical Genetics

among the 5000 listed genetic diseases, over 1100 genes can be tested clinically

half of the 2% of fetuses with major genetic abnormalities have abnormal chromosomes

30% of children admitted to the hosphital have a genetically related problems

Centromere Location

metacentric: when centromere is in the center

submetacentric: when centromere is closer to one side then the other (but both still the same length)

acrocentric: when the centromere is at one end and one is sticks out a little farther then the other one

p arm is the short arm, q arm is the long arm

Banding patterns

can depend on different ratios of the basepairs

if stained with an A-T binding protein then parts of a chromosome would look brighter if it had A-T and dimmer if it had G-C

when fluorescing basepairs, the more in a row, the more exponentially bright it is(2 – 4x brightness, 3- 9x brightness)

Acrocentric chromosomes

contain stalks and satellites

about 10 chromosomes like this

ribosomal genes tend to be on stalks

Down Syndrome from Trisomy 21

palpebral fissures are slanted upward and outward

flat nasal bridge

round face

small mouth, thick lips, often large tongue

average IQ of 50 at 5 years, 38 at 15 years and later degenrates further

hypotonic infants (floppy)

Nondirective counseling

provides all known information and allows the patient to decide

couple informed of fetal status, advised of the possibilities, and then advised to decide without the help of a counselor or physician but with trusted individuals and families with similarly affected patients

Molecular Cytogenetics

can test chromosome copy number and single gene copy number

enabled by cloned genes and other DNA fragments that can be sequenced, labeled with specific fluorescent colors, and hydridized specifically to similar or identical DNA sequences on chromosome bands, and viewed at 1000-fold magnification

Microarray Principles

microarrays test hundres to many thousands of specific chromosome targets simultaneously by labeling total DNA from a patient sample in the first color and total DNA from normal DNA in the second color and hybridizing these to all of the specific cloned genomic sites bound to previously designated locations on the microarray

comparison of both fluorescent colored signals at each location determines whether the patients DNA has less, the same, or more copies of the DNA target than normal DNA. many locations to small to be seen on banding or FISH can be tested at the same time to look for abnormal gene copy numbers in doezens of gene disorders as well as abnormal chromosome locations

FISH is the clinically validated test used to confirm abnormal microarray results

Retinoblastoma

may be inherited or arise following two gene mutations to both retinoblastoma genes in the same cell

used to pluck out the eye

looks like white spot in eye

prenatal diagnosis, routine eye exam

laser beam destroys early tumors

inherit one bad gene, and mutation causes other, or two bad mutations

Other approaches to

Treating genetic diseases

3. transplantation(adult polycystic kidney disease)

4. modifying diet (maternal PKU)

5. Gene therapy(adenosine deaminase)

Enabling PCR Attributes

primer selection: four different basepair possibilities at each of 17 locations